Name: rapid identification of chemical genetic interactions in saccharomyces cerevisiae
Uploaded: 2015-04-05T14:00:00.000+00:00
Duration: 12 min 13 s
Description: Watch this Scientific Journal Video about Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae at JoVE.com

研究在CJN实验室由NSERC，加拿大癌症协会研究所（CCSRI），以及加拿大乳腺癌基金会（BC-育空分公司）工作的资助。

注意:这个程序的一般工作流程是在图2列出。 1.测定生长抑制的剂量<ol><li>酵母过夜培养的制备 注:在本协议中所使用的酵母提取物，蛋白胨-葡萄糖培养基（YEPD）是一个标准的配方13。 <ol><li>条纹出BY4741（ 的MATa HIS3Δ1leu2Δ0met15Δ0ura3Δ0）细胞在YEPD琼脂平板孵育48小时，在30℃，或直至可见菌落形成。 </li><li>接种5毫升YEPD液体介质中的无菌培养容器与单个菌落制备过夜培养物。隔夜孵育在30℃，连续旋转或晃动。培养通常由早上（〜2×10 8个细胞/ ml）达到饱和。 </li></ol></li><li>固体琼脂培养基制备含有不同的化学剂量<ol><li>准备几个股票化学将在100倍所需的终浓度在适当溶剂中进行测试。 注:有效浓度将取决于化学，并且必须凭经验确定。因此，建议最初测试了广阔的终浓度范围内， 即从低μM到高的MM。 </li><li>对于化学品的每种浓度进行测试，等分3毫升熔融YEPD琼脂到几个无菌培养管中。发生在55℃水浴，以防止凝固。 </li><li>对于化学的各个浓度待测试添加30微升的100倍的化合物的熔融媒体，涡旋2-3秒，以混合，并且吸管1毫升成每对重复试验孔在12孔板中。一定要包括车辆只能控制。允许板过夜设定为室温。放弃任何额外介质。 </li></ol></li><li>传播电镀细胞<ol><li>接种10毫升YEPD液体介质与2微升饱和酵母培养物，一夜之间成长为在步骤1中。1.2。 </li><li>板25微升稀释酵母培养物每孔，均匀涂抹用无菌玻璃珠或杆，并允许板下火焰干燥。孵育板在30℃下48小时。 </li><li>评价酵母在平板上生长（菌落两者总数和大小）。选择的化合物的亚致死浓度不通过大于10％的抑制生长15％来执行屏幕。 </li></ol></li></ol> 2.系统化学遗传屏幕<ol><li>缺失突变体阵列的维护源板（DMA）和制备<ol><li>有关从甘油股票构建缺失突变体阵列的详细说明请参考Baryshnikova 等 14。一旦缺失突变体已被排列，以每板384菌落的密度时，DMA可以在4℃保存数月。复制在含有作为必要的G418的200微克/毫升，以防止从菌落生长到每个YEPD琼脂等。 注意:多个串行复制应该避免，以防止生长缓慢的品系的损失最小化和抑制的突变体的外观。这一点尤其重要，如果保持藏品的单倍体。 </li><li>执行使用微生物排列机器人系统中的所有副本电镀步骤。另外，操纵阵列使用手动钉扎工具殖民地。 注:酵母删除集合可从几个商业渠道单倍体和二倍体和通常运作为甘油储在96孔板中。 </li></ol></li><li>冷凝的缺失突变体阵列<ol><li>制备250毫升含有G418的200微克/毫升YEPD琼脂介质。 G418的有效浓度可以变化，并且每批应凭经验测试。 </li><li>通过倾倒50毫升含有每板的G418的200微克/毫升YEPD琼脂制备5 YEPD琼脂平板上。冷凝前阵做到这一天，让板冷却，在室温erature隔夜放在平坦的表面上。 注:四板将需要采集在1536株密度，第五板倾作为额外。 </li><li>除去含有以每板384菌落密度的突变体缺失集合16的培养皿，并允许达到室温（约1小时）。 </li><li>擦拭用一个棘手的任务擦拭已形成于各板的盖子的任何冷凝。如果不这样做，可能会导致水滴沉积的阵列和阵列突变体的交叉污染上。 </li><li>使用微生物阵牵制机器人，从384菌落每板（16培养皿）至1536菌落每盘（4培养皿）的密度凝聚DMA。执行此以便从板1引脚的1次菌落到行冷凝的阵列的第1列1，板2的1次菌落到行1列2，板3的第1次菌落到行2和列1，和板4的第一届殖民地行2第2列。 </li><li>孵育统一阵列在30℃下过夜。 </li><li>检查阵列，以确保殖民地从源盘均匀传输。会有若干空白位置，故意掺入到所述阵列，其中作为指导，以确保在DMA已经凝结正确（ 图4A）。 注意:这些将是复制品镀源板​​在含有试验化合物的介质。单个源板可用于多种pinnings。也有不少机器人将有一个对冲的功能，这将确保酵母正在每次传输类似的数字。 </li></ol></li><li>副本板缺失突变体阵列<ol><li>准备700毫升管制（车辆专用）和700毫升实验媒体（含化学品）的。这是足够用于进行一式三份的测定（每个条件12板，加上两个额外）。 </li><li>倒入50毫升YEPD琼脂含测试化学或每盘的控制。允许板吨Ø在室温下冷却过夜平坦的表面上。 </li><li>使用机器人复制品板，接种在DMA上三套含有化学品的试验确定浓度的板，以及三组含的车辆控制的板。孵育板在室温下搅拌24-48小时。 </li></ol></li></ol> 3.成像板和数据分析<ol><li>从孵化器中删除板，并允许他们来室温（约1小时）之前的成像。这将防止凝结而成像板形成。 </li><li>在24和48小时通过移除盖和放置面朝下放在平板扫描仪图像板。在至少300 dpi的分辨率捕捉图像。另外，使用数码相机拍摄的图像。如果这样做，地方板块面朝上放在一个黑暗的背景，取下盖子图像。 注:板的文件格式和定位将取决于所使用的量化方案。 </li&gt; <li>进行量化，用几个开源项目，包括Balony 15，SGAtools 16和ScreenMill 17的一个阵列殖民地大小的比较。 </li></ol> 4.验证屏注:在这个阶段，几个酵母突变体的缺失将比分为过敏感兴趣的化学品。这些化学遗传相互作用必须接下来在两个方面进行验证。第一敏感菌株的身份应通过PCR来确认。二，化学敏感度必须独立评分。下面概述是一个简要的协议用于进行斑点试验，以验证应变超敏反应，在酵母中的生物学共同的技术。 <ol><li>条纹出所需的（过敏性）从酵母缺失突变体集合到含有G418的200微克/毫升YEPD琼脂上。孵育在30℃，直至菌落形成。通过诊断性PCR验证菌株的基因型与根据引制造商的协议。 </li><li>接种将5ml YEPD液体每个菌株的孵育过夜，在30℃下用旋转或摇动。 </li><li>测量OD 600和冲淡每个菌株以OD 600 = 1在无菌的dh 2 O.这些是目前的标准化的酵母培养物。 </li><li>分配100μl的归一化的野生型酵母（BY4741）培养至A1孔96孔板。分配100μl的附加多达七个品系到孔B1-H1。 </li><li>使用多通道移液器通过A6-H6免除90微升无菌的dh 2 O的水井A2-H2。最后，准备了一系列1:10稀释归酵母培养的。开始由串联吹打10微升塔1至第2列与多通道移液器，并继续在96孔板直到6稀释液制成（ 即 10 0到10 -5）。 </li><li>使用多通道传输2-5微升稀释的酵母培养物中的网格移液器到含有化学和车辆控制YEPD固体培养基。孵育板在适当温度下24-48小时。 </li><li>检查板和比较选择的突变体，以化学（相对于BY4741控制）的灵敏度。在24和图像板48小时如在步骤3.2。 注意:虽然几种过敏反应菌株与相关基因本体或功能的识别借给信心到屏幕的有效性，一种过敏反应缺失菌株用含有该基因的野生型拷贝的质粒的转化是必需的正式建立一个基因删除的利息（而不是隐藏第二位点突变）导致药物的敏感性。 </li></ol>

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The authors declare they have no competing financial interests.

这里列出的是产生化学遗传相互作用的配置文件的方法。该方法是简单的:通过比较在对化学品的存在和不存在完整基因缺失集合各菌株的菌落的大小，要容忍化学损伤所需的所有基因被识别。所得敏感菌株的列表的注释富集分析然后可以用于提供洞察行动的化学物质的模式。虽然该协议已被优化的芽殖酵母，它也可以适用于与其他阵列微生物缺失的集合，如大肠杆菌使用大肠杆菌庆...

的遗传工具，并在模式生物酿酒酵母可利用的资源已启用大规模功能基因组学的研究，它们共同提供了新的见解的基因如何充当网络来履行生物系统的要求。这些工具的基础是在酵母1,2-一套完整的所有开放读框的非必需基因缺失的协作创作。一个显着的观察是，只有约20％的酵母基因时，作为标准的实验室条件下培养单倍体所需的可行性。这凸显了一个细胞通过替代生物途径的利用率，减缓基因组扰动的能力。遗传突变体是可行的单独的，但致命的组合，信号连接或收敛平行生物途径和形式描述的生物学功能的基因相互作用网络。带有条件TE的发展必需基因的mperature敏感和亚效等位基因的等位基因的技术还没有被限制在非必需基因3,4的研究。这个概念已经在基因组规模已经应用产生偏见的遗传相互作用地图说明如何参与类似的细胞过程的基因聚集在一起5。 基因网络的化学扰动模拟基因缺失（ 图1）6。查询生长抑制化合物对缺失株的高密度阵列，用于超敏反应标识化学遗传相互作用信息， 即所需要容忍化学应力的基因的列表。像遗传相互作用，化学文库的大规模的屏幕显示，与动作一起簇7的类似模式的化合物。因此，通过建立一个化合物的化学遗传相互作用轮廓的作用方式，可以通过将其与LAR比较推断GE规模合成的遗传和化学遗传相互作用数据集8,9。 大型化学遗传筛选，其中化合物的分数审问，已经由条码竞争测定进行。在这种方法中，缺失菌株的汇总收集于小体积的含有化学介质中生长集体几代。由于每个缺失突变窝藏一个独特的遗传条形码，个别突变体缺失菌株的池内的生存能力/生长被微阵列或高通量测序10跟踪。 通过监测生长在含有生物活性化合物的固体琼脂物理阵列突变体菌落大小推断健身也识别化学遗传相互作用11,12的有效方法。这种方法提供了一种具有成本效益的替代竞争为基础的筛选，非常适合用于测定化学品的小库。这里所概述是用于制造在酿酒化学遗传相互作用的列表的简单方法酵母不依赖于分子生物学操作或基础设施，它仅需要一个酵母删除集合，机器人或手动钉扎装置中，并自由可用的图像分析软件。

<table><tbody><tr><td>Yeast Extract</td><td>BioBasic</td><td>G0961</td><td>For YEPD liquid/solid media add to 1% final concentration (w/v)</td></tr><tr><td>Tyrptone Powder</td><td>BD Biosciences</td><td>211820</td><td>For YEPD liquid/solid media add to 2% final concentration (w/v)</td></tr><tr><td>Dextrose</td><td>Anachemia</td><td>31096-380</td><td>For YEPD liquid/solid media add to 2% final concentration (w/v) &amp;#8212; do not autoclave. Prepare 20% stock solution, filter sterilize, and add to media after autoclaving.</td></tr><tr><td>Agar A</td><td>Bio Basic</td><td>FB0010</td><td>For YEPD solid media add to 2% final concentration (w/v)</td></tr><tr><td>G418</td><td>A.G. Scientific Inc.</td><td>G-1033</td><td>Prepare 1,000x stock at 200&amp;nbsp;mg/ml in dH&lt;sub&gt;2&lt;/sub&gt;O and filter sterilize.&amp;nbsp;</td></tr><tr><td>12-well plate</td><td>Greiner Bio One</td><td>655180</td><td></td></tr><tr><td>5 ml culture tubes</td><td>Evergreen Scientific</td><td>222-2376-080</td><td></td></tr><tr><td>10 cm Petri Dish</td><td>VWR</td><td>25384-302</td><td></td></tr><tr><td>ROTOR HDA</td><td>Singer Instruments&amp;nbsp;</td><td>ROT-001</td><td>high-throughput microbial array pinning robot</td></tr><tr><td>PLUSPLATE&amp;copy; Petri Dish</td><td>Singer Instruments&amp;nbsp;</td><td>PLU-001</td><td>Box of 200 dishes</td></tr><tr><td>384 Short-Pin RePad</td><td>Singer Instruments&amp;nbsp;</td><td>RP-MP-384</td><td>Box of 1,000 pads</td></tr><tr><td>1536 Short-Pin RePad</td><td>Singer Instruments&amp;nbsp;</td><td>RP-MP-1536</td><td>Box of 1,000 pads</td></tr><tr><td colspan="4">Alternative Pinning Tools:</td></tr><tr><td>Fully Automated Robtic Systems</td><td>S&amp;amp;P robotics</td><td>http://www.sprobotics.com</td><td>Several automated colony handling robitic and imagining systems available.</td></tr><tr><td>Manual Pinning Tools</td><td>V&amp;amp;P Scientific</td><td>http://www.vp-scientific.com</td><td>Handheld replication tools and accessories.&amp;nbsp;</td></tr></tbody></table>

rapid identification of chemical genetic interactions in saccharomyces cerevisiae

确定生物活性的化学物质的作用方式是感兴趣的范围广泛的学术，医药，和工业的科学家。 酿酒酵母，或出芽酵母，是一个模型真核生物的量的〜6000基因缺失突变体和亚效等位基因必需基因的完整集合突变体是市售的。突变体的这些集合可以被用来系统地检测化学基因的相互作用，必要即基因容忍的化学物质。这个信息，反过来，报告了化合物的作用可能模式。在这里，我们描述了一个协议，用于快速识别在芽殖酵母化学基因的相互作用。我们证明使用化疗剂5-氟尿嘧啶（5-FU），其具有定义良好的作用机制的方法。我们的研究结果表明，都需要在5-FU的存在下，这是与以往的米一致增殖外来体和DNA修复酶的核TRAMP核糖核酸icroarray基于条形码化学遗传方法和5-FU的RNA和DNA代谢产生不利影响的认识。这些高通量筛选所需的验证方案也有所说明。

作为这种方法的一个验证我们进行化疗剂5-氟尿嘧啶（5-FU），按照以上概述的协议的一个代表性化学遗传相互作用屏幕。 5-FU是已知破坏胸苷酸合成酶，以及DNA和RNA代谢18。 5-FU的化学遗传相互作用是很好的研究，并已研究了通过使用两个杂合和纯合缺失集合8,19酵母条形码微阵列技术。在这里，我们表明，类似的结果可以通过突变菌落大小比较定量来获得。 应...

Watch this Scientific Journal Video about Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae at JoVE.com

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

化学遗传相互作用的快速鉴定<em&gt;酿酒酵母</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

rapid-identification-chemical-genetic-interactions-saccharomyces

Research

JoVE Journal

Biology

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

10.3K 次观看.  University of Victoria. Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

视频: Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

科研

遗传学

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

一种在裂变酵母中的深度固序辅助自发抑制屏

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

通过全基因组互惠性分析,对糖母菌菌种间热耐性差异的遗传图谱

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

生物学

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

通过邻近连接和定量PCR检测酿酒酵母同源重组中间体

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

格拉菲克斯用于检测 糖精 的瞬态相互作用器

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

生物化学

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

微阵列分析FOR酿酒酵母</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

成长型测定和遗传需求生化确认的蛋白质降解<em&gt;酿酒酵母</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype.
This paper describes a protocol for carrying out this &#39;multicopy suppression&#39;-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

用于鉴定裂殖酵母多拷贝抑制因子的遗传筛选

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships ...

化学遗传相互作用的快速鉴定

摘要

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与ChEC-seq的蛋白质-DNA相互作用的全基因组映射

一种在裂变酵母中的深度固序辅助自发抑制屏

通过全基因组互惠性分析,对糖母菌菌种间热耐性差异的遗传图谱

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

通过邻近连接和定量PCR检测酿酒酵母同源重组中间体

格拉菲克斯用于检测糖精的瞬态相互作用器

微阵列分析FOR酿酒酵母

条形码酵母图书馆竞争力的基因屏幕

成长型测定和遗传需求生化确认的蛋白质降解

用于鉴定裂殖酵母多拷贝抑制因子的遗传筛选

化学遗传相互作用的快速鉴定

摘要

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此视频中的章节

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射

一种在裂变酵母中的深度固序辅助自发抑制屏

通过全基因组互惠性分析,对糖母菌菌种间热耐性差异的遗传图谱

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

通过邻近连接和定量PCR检测酿酒酵母同源重组中间体

格拉菲克斯用于检测 糖精 的瞬态相互作用器

微阵列分析FOR酿酒酵母

条形码酵母图书馆竞争力的基因屏幕

成长型测定和遗传需求生化确认的蛋白质降解

用于鉴定裂殖酵母多拷贝抑制因子的遗传筛选

格拉菲克斯用于检测糖精的瞬态相互作用器